international journal of pharma and bio sciences issn 0975 ... · source for biomaterials such as...

Int J Pharm Bio Sci 2015 July; 6(3): (P) 162 - 178

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Review Article Pharmaceutics

International Journal of Pharma and Bio Sciences ISSN

0975-6299

A REVIEW ON BIOMEDICAL APPLICATIONS OF

CHITOSAN-BASED BIOMATERIALS

MASAYUKI ISHIHARA*, HIDEMI HATTORI AND SHINGO NAKAMURA

Research Institute, National Defense Medical College, 3-2 Namiki,

Tokorozawa, Saitama 359-8513, Japan.

ABSTRACT

Chitin/chitosan and their derivatives have attracted considerable interest as a potential

source for biomaterials such as hydrogels due to their safety and biological activities,

such as, antimicrobial, antitumor and stimulation of wound healing, etc. In particular,

some kinds of covalently cross-linked (chemical) chitosan hydrogel such as chemically

cross-linked chitosan hydrogel, photocrosslinked chitosan hydrogel (PCH) and ionic

crosslinked (physical) chitosan hydrogels such as ionic/temperature sensitive chitosan

hydrogel and polyelectrolyte complexes (PECs) composing positive or negative charge

have been developed. These have been used in several applications including drug

delivery carriers, hemostats, wound dressings, submucosal fluid cushion, tissue

adhesive and scaffolds of tissue engineering which we originally evaluated. In this review,

we described on chitosan hydrogels with particular attention on medical applications of

PCH, hydrocolloids and PECs in fields of Biomedical Research.

KEYWARDS: Cross-linked Chitosan Hydrogel, Polyelectrolyte Complexes, Drug Delivery Carriers,

Hematostats, Tissue Adhesive, Wound Dressing.

*Corresponding author

MASAYUKI ISHIHARA

Research Institute, National Defense Medical College, 3-2 Namiki,

Tokorozawa, Saitama 359-8513, Japan.



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INTRODUCTION

Chitin/chitosan can be produced

economically from the shells of crustaceans,

a waste product of the seafood industry that

would otherwise pollute coastal areas.

Chitosan comprises co-polymers of

N-acetyl-glucosamine and N-glucosamine

units linked by β-(1→4) glycosidic bonds, and

can be obtained by alkaline deacetylation of

chitin1,2. Chitosan is nontoxic and

biocompatible with living tissue3,4. The

production of chitin from shells mainly

involves the removal of proteins and the

dissolution of calcium carbonate in the shells.

The resulting chitin is deacetylated to yield

chitosan1,2. The term “chitosan” is used to

describe polymers comprising less than 50%

N-acetylglucosamine units2-4. The degree of

deacetylation (DDAc) affects the solubility,

hydrophobicity and electrostatic properties of

chitosan, with the latter affecting the

polymer’s ability to interact with polyanions

through the protonated amino groups.

Chitosan can be hydrolyzed by lysozyme and

is thus a biodegradable polymer. Chitosan

and its degradation products are nontoxic,

nonimunogenic and noncarcinogenic3-6.

Furthermore, chitin/chitosan and their

derivatives have attracted considerable

interest due to their biological activities,

including antimicrobial7, hypocholesterolemic

functions8, antitumor9,10 and their stimulation

of wound healing11,12. The present review is

exclusively concerned with chemical chitosan

hydrogels formed by addition of a

crosslinker13, namely covalently crosslinked

such as photocrosslinked chitosan hydrogels

(PCH) formed by addition of a

photocrosslinker14-16. A second review will

describe physically cross-linked chitosan

hydrogel such as temperature sensitive

chitosan hydrogel17,18, polyelectrolyte

complexes (PECs)19,20 and hydrocolloid21,22

formed by direct interaction between

polymeric chains without the addition of cross

linkers. An entangled chitosan hydrogels

which are formed by solubilization of chitosan

in an acidic aqueous medium will not be

discussed further in this review, as they are

limited by their lack of mechanical strength

and their tendency to dissolve. In the present

review articles, we focus the potential

medical applications of photocrosslinked

chitosan hydrogel (PCH)14-16 and

chitosan-based biomaterials such as

hydrocolloid sheets composed of alginate

chitin/chitosan, fucoidan hydrocolloid sheet

(ACF-HS)21,22 and polyelectrolyte complexes

(PECs) composing chitosan and

protein/gene19 which we had originally

evaluated, as drug delivery carriers, tissue

adhesives, submucosal fluid cushion, wound

dressing, hematostats, scaffolds for tissue

engineering and protein/gene delivery

carriers.

Chitosan-based hydrogels

Chitosan hydrogel was defined as

macromolecular networks swollen in water or

biological fluids. Based on the definition given

here, chitosan hydrogels are often divided

into two classes, namely chemical hydrogels

and physical hydrogels13,23,24. Chemical

hydrogels are formed by irreversible covalent

links, as in covalently crosslinked chitosan

hydrogels. On the other hand, physical

hydrogels are formed by various reversible

links. These can be ionic interactions as in

ionically cross-linked and PECs, or



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secondary interactions such as

alginate/chitosan/fucoidan complexed

hydrocolloid sheets (ACF-HS)21,22. The

present review is exclusively concerned with

chitosan hydrogels formed by addition of a

crosslinker, namely covalently or ionically

crosslinked hydrogel. In cross-linked

hydrogels, polymeric chains are

interconnected by crosslinkers, leading to the

formation of a 3D network. Crosslinkers are

molecules of molecular weight (MW) much

smaller than the MW of the polymeric chains.

The properties of cross-linked hydrogels

depend mainly on their crosslinking density,

namely the ratio of moles of crosslinking

agent to the moles of polymer repeating

units23,24. Figure 1A shows simplified

scheme for temperature sensitive chitosan

hydrogel which show sol-gel transition at

body temperature due to a conformational

change. Since chitosan lucks intrinsic

thermosensitive properties, other

temperature sensitive materials need to be

introduced into the chitosan to make it

applicable as a temperature sensitive

chitosan hydrogel. For example, temperature

sensitive hydrogels composed of chitosan

and β-glycerophosphate (GP)13,18 or

polyethylene glycol (PEG)13 has been

prepared and investigated their sol-gel

transition in response to thermal and pH

changes. Those hydrogels has also been

evaluated as carriers for cells and

drug-delivery25,26. Preparation of a hydrogel

containing a covalently cross-linked chitosan

hydrogel requires crosslinkers which are

molecules with at least two reactive

functional groups that allow the formation of

bridges between chitosan chains (Figure 1B).

Those crosslinkers should have at least two

reactive functional groups that allow the

formation of bridges between polymeric

chains such as glutaraldehyde as

dialdehydes13. However, even if hydrogels

are purified before administration, the

presence of free unreacted dialdehydes in

hydrogels could not be completely excluded

and may induce toxic effects. Figure 1C

shows simplified scheme for PCH which form

hydrogels under short exposure to visible or

ultraviolet (UV) light in the presence of light

sensitive compounds (photocrosslinkers)14-16

.



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Figure 1

Simplified scheme of gelling mechanism.

A: Thermal gelation due to a change in

temperature the polymer molecules

rearrange from random coil to helix, then the

helices assemble in dusters join together. B:

Chemical crossling gelation due to chemical

reaction between crosslinkers and polymers.

C: Photocrosslinking due to radical reaction

between photocrosslinkers and polymers.

We previously described a

photocrosslinkable chitosan derivative

(Az-CH-LA) that contains both lactose

moieties (lactobionic acid) and photoreactive

azide groups (p-azidebenzoic acid) as

photocrosslinker14-16. The chitosan used in

this study had a molecular weight of 300－

500 kDa with 80% deacetylation. Lactose

moieties have been introduced through

condensation reactions of chitosan with

amino groups. Moreover, chitosan containing

2% lactobionic acid exhibited high aqueous

solubility, even at neutral pH. Furthermore,

application of ultraviolet light (UV) irradiation

with a 250-W lamp (major peak, 340 nm;

Usio Electrics Co., Ltd., Tokyo, Japan) to

Az-CH-LA produced an insoluble PCH just

like soft rubber within 30 seconds and firmly

adhered two pieces of ham to each other14-16.

The Az-CH-LA solution can be injected into

body and the hydrogels are then formed by

applying UV light externally through skin.

Basic molecules such as chitosan and

protamine complexed with acidic molecules

such as alginate, heparin and fucoidan form

complexes through ionic interactions as

PECs13,24,26. Reported studies indicate that

polyanions and polycations can bind to

proteins below and above their isoelectric

points, respectively. These interactions can

result in nanoparticles, hydrogels, soluble

complexes and/or the formation of



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amorphous precipitates (Figure 2). Main

aspects studied by different authors are

compositions of PECs obtained under

various experimental conditions, such as the

strength and position of ionic sites, charge

density and rigidity of polymer chains as well

as chemical properties such as solubility, pH,

temperature and concentration13,26.

Electrostatic interactions are also important

because of their similarity to biological

interactions. Interactions between proteins

and nucleic acids, for example, play a role in

the transcription process27. DNA/chitosan

PECs28, chitosan/chondroitin sulfate PECs

and chitosan/hyaluronate PECs function as

gene29 and drug carriers29. Moreover, PECs

that are insoluble also have potential

applications as membranes, microcapsules,

micro/nanoparticles and scaffolds for tissue

engineering29.

Figure 2

Formations of PECs such as nanoparticles and hydrogels.

Biological adhesives, hemostats and

submucosal fluid cushion

Biological adhesive are used for tissue

adhesive, hemostasis and sealing of the

leakage of air and body fluids during surgical

procedure. Although most bleeding in

surgical procedures can be controlled by

appropriate sutures, hemostasis is

uncontrollable under certain conditions, such

as coagulopathy, medication of

anticoagulants, inflammation, infection and

severe adhesion14,15. In addition, intractable

air leakage in lung surgery has often been

found, especially in emphysematous lung



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disease15. In many cases of such

uncontrollable bleedings and intractable air

leakages, a number of adhesives have been

utilized in hemostasis and air sealing, i.e.

chemically crosslinkable gelatins30,

cyanoacrylate polymers31,32 and fibrin

glues33,34. Requirements for such adhesives

are locally non-irritating, systematically

nontoxic, appropriately flexible and

biodegradable. However, cytotoxicity and

severe tissue irritability have been found

when using resorcinol, formaldehyde or

carbodiimides for the crosslink-reaction of

gelatins30 or due to the formation of

formaldehyde by degradation of

cyanoacrylate31,32. Fibrin glue, which

contains fibrinogen, thrombin, factor XIII and

a protease inhibitor, utilizes the blood

coagulation system for sealing tissues and

currently is the most widely used surgical

adhesive33,34. However, fibrin glue has a

disadvantage in its industrial production,

since human blood is used as its source. On

the other hand, curable chitosan-poly

(ethylene glycol)-tyramine hydrogels35,

catechol-functionalized chitosan/pluronic

hydrogels36 and PCH14,15 were reported as

chitosan-based hydrogels for tissue adhesive.

The binding and sealing strengths of the PCH

prepared from 2 w% Az-CH-LA solution was

superior to that of fibrin glue (Beriplast P)14,15.

A tracheal tube was inserted into the dead pig

and connected to a mechanical ventilator.

The lung was then punctured with a needle

(1.2 mm in diameter) about 10 mm deep.

One drop (about 30 µL) of 30 mg/mL of

Az-CH-LA solution was applied to the

puncture site and irradiated with UV light at a

distance of 2 cm for 30 seconds.

Subsequently, ventilation was started through

a linear pulsed-air volume increase. The

pressure at which air leakage reoccurred was

measured and termed the “bursting pressure”

of the PCH (millimeter of mercury)14,15.

Beriplast P was also examined as a control

and was measured for the bursting pressure

occurred 5 minutes after application of the

fibrin glue. On the other hand, one end of

small intestine, trachea and thoracic aorta

removed from the dead pigs was ligated with

suture material, and the other side was

intubated with a small catheter held in place

by ligature. The catheter was connected to a

syringe and a manometer. The issues were

punctured with the needle, and about 30 µL

of the Az-CH-LA solution was applied to the

hole and irradiated with UV light for 30

seconds. The tissues were placed under

water, and they were inflated until leakage

bubbles could be detected in the water. The

pressure required to produce this air leakage

was measured as the bursting pressure.

Similar experiments have been performed

with the fibrin glue, with the bursting pressure

measurements starting 5 minutes after

application14,15. Results of above

experiments were shown in Table 115. The

bursting pressures of PCH were more than

that of the fibrin glue on lung, small intestine,

trachea and thoracic aorta. These results

suggest that the sealing strength of PCH may

be sufficient to stop arterial bleeding and air

leakage from the lung or trachea in surgical

applications.



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Table 1

Air-sealing strength of chitosan hydrogel and fibrin glue15

Sealing strength (mmHg)

Organs Chitosan hydrogel (PCH) Fibrin glue

(TC)

Lung 51 ± 11 (n = 4) 12 ± 2 (n = 4)

Small intestine 65 ± 5 (n = 6) 48 ± 7 (n =

6)

Trachea 77 ± 29 (n = 6) 44 ± 16 (n =

6)

Thoracic aorta 225 ± 25 (n = 6) 65 ± 15 (n =

6)

We examined the hemostatic efficacy of

photocrosslinkable chitosan hydrogel-mixed

photocrosslinked chitosan sponges (PCM-S)

(Figure 3) after hepatic injury of rats37. The

left lobe of the liver was penetrated with a

dermal punch to produce a penetrating

wound in heparinized rats. Treated rats either

had PCM-S applied into the wound and then

were immediately UV-irradiated, or they had

TachoComb® (TC) inserted into the wound38.

The study demonstrated that PCM-S

effectively controlled hemorrhage after liver

trauma in the heparinized rats. All

heparinized rats in PCM-S-treated groups

achieved complete hemostasis within 5

minutes and all survived. In contrast, the

control heparinized rats could not stop the

bleeding for more than 3 hours and all rats

were died within 6 hours. TC had an

intermediate effectiveness, with bleeding

lasts longer than 20 minutes, resulting in

three deaths of three of the eight study rats

during the first 24 hours. No adverse events

related to the use of the hemostatic agents

(PCM-S and TC) were detected through two

months in both non-heparinized and

heparinized rats37. Furthermore, A novel

emergency hemostatic kit was developed for

severe hemorrhage using PCH39.



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Figure 3

Formation of photocrosslinkable chitosan (A)-mixed photocrosslinked chitosan sponge (C) (PCM-S). Az-CH-LA is converted to photocrosslinked chitosan hydrogel (B) with UV irradiation.

Formation of a submucosal fluid cushion

(SFC) has become integral to endoscopic

endoscopic submucosal dissection (ESD) as

well as endoscopic mucosal resection EMR

of large superficial lesions of the

gastrointestinal tract40. We also investigated

the use of PCH as SFC41-43. A disadvantage

of PCH-assisted ESD is to require UV

irradiation using an expensive UV fiber for

ESD, which may be associated with minor

inflammation in residual tissues42,43.

Furthermore, it cannot be ruled out that

PCH-assisted ESD may have an association

with carcinogenesis. Because homogenous

UV irradiation using a simple UV lamp and

fiber is technically difficult, further studies are

necessary to determine the requirement and

safety of UV irradiation42,43. Since those

biomaterials as SFC were hard to inject

because of their high viscosity, an application

of a targeted high-pressure water jet may be

required to ameliorate the endoscopic

treatment of mucosal lesion44. The

application of an ideal injectable hydrogel as

SFC among those hydrogel described in this

review could contribute to ameliorate the

endoscopic treatment which previously could

not be resected endoscopically due to their

size, extent or location.

Chitosan in regenerative medicine

Regenerative medicine, one of the hottest

fields in present and future life science, finally

aims at the restoration or replacement of lost

or damaged organ or body part with

transplantation of new tissues in combination

with supportive scaffolds and biomolecules.

Recently, functional biomaterial research has

been directed toward the development of

improved scaffolds for tissue engineering3,4,23,

wound dressing12,16,45 and drug delivery

carrier5,6. In this regard, increasing attention

has been given to chitosan and its derivatives.

Chitosan and its derivatives are undisputed



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biomolecules of great potential by their

polyelectrolyte properties, including the

presence of reactive functional groups,

gel-foaming ability, high adsorption capacity,

biodegradability, bacteriostatic, fungistatic

and even anti-tumor influence4,7,10. Several

requirements have been identified as crucial

for the production of tissue engineering

scaffolds: (1) the scaffold should be made

from material with controlled biodegradability

or bioresorbability so that tissue will

eventually replace the scaffold, (2) possess

interconnecting pores of appropriate scale to

favor tissue integration and vascularization,

(3) have appropriate surface chemistry to

favor cellular attachment, differentiation and

proliferation, (4) possess adequate

mechanical properties to match the intended

site of implantation and handling, (5) should

not induce any adverse response and (6) be

easily fabricated into a variety of shapes and

size46,47. The versatility of chitosan and its

derivatives offer a wide range of applications

since they are biodegradable and nontoxic,

and can be formulated in a variety of forms

including powders, gels, membranes,

sponges and films for their applications. They

can also provide controlled release of growth

factors and extracellular matrix components.

However, unfortunately chitosan alone

cannot meet the long-term mechanical,

geometrical, functional and cell adherent

requirements46,47. To improve the adherent

ability for seeding cells, the chitosan allow for

wide range of molecules to be modified. The

incorporation of collagen or biologically active

RGD-containing protein peptides to chitosan

as a chitosan-collagen scaffold can enhance

its cell attachment ability46,47. Table 2

summarized on applications and benefits of

chitosan in regenerative medicine including

wound healing, tissue engineering and drug

delivery.

Table 2 Application and benefits of chitosan in regenerative medicine.



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There are many other synthetic materials which

can react biocompatibly with the body. Among

these materials, polylactide (PLA), polyglycolide

(PGA) and polylactide-co-glicolide (PLGA) etc.

have received much attention because of their

biodegradability and biocompatibility.

Conjugation of chitosan with those synthetic

materials is expected to become a key and

potential technology to develop desirable

scaffold materials for the tissue regenerations48.

Stem cells with self-renewal potential and

multilineage differentiation capacity have been

in tissue engineering49. Bone marrow- or

adipose tissue-derived mesenchymal stem cells

have been extensively studied and have shown

promising application implication50. Furthermore,

recent studies show that chitosan has good

characteristics for the attachment, proliferation

and viability of mesenchymal stem cells50. With

these promising features, they are considered

as an interesting biomaterial for use in cell

transplantation and tissue regeneration, and the

technology for chitosan has been used to create

various tissue analogs including cartilage51,

bone52, skin53, myocardium54 and peripheral

nerve55 in the past decades.

Chitosan in wound healing

Chitosan possesses the characteristics

favorable for promoting rapid dermal

regeneration and accelerated wound healing. It

is observed that chitosan has a stimulatory

effect on macrophages and that it was found to

act as chemoattractant for neutrophils both in

vitro and in vivo, an early event essential in

wound healing53. These cells kill

microorganisms, remove dead cells and

stimulate the other immune system cells, which

improve overall healing by reducing the

opportunity for infection56. The application of

chitosan and its derivatives as a wound

dressing has been widely studied. In a

comparative study of insoluble chitin powder,

insoluble chitosan powder and water-soluble

chitin/chitosan (WSC) solution, WSC solution

was found to have the highest tensile strength

with the fastest healing rate57. It is likely that the

superior biodegradability and hydrophilicity of

WSC solution can enhance its compatibility with

wounded tissues and increase its activity as a

wound-healing accelerator57. To improve the

healing process, chitosan has been combined

with a variety of functional molecules such as

growth factors, extracellular matrix components

and antibacterial agents. The other advantages

include healing of wounded meniscal tissues,

and of decubitus ulcers, depression of capsule

formation around prostheses, limitation of scar

formation and retraction during healing57. We

have previously reported that the application of

PCH into open wounds induces a significant

wound contraction, thereby accelerating the

wound closure and healing process, as shown

in a normal mouse45 or rat12 model for wound

repair. In addition, the PCH showed the ability to

control release of various growth factors, to serv

as a novel carrier and to induce

neovascularization in vivo. FGF-2 interacted

with PCH and the FGF-2 molecules

incorporated into the PCH were gradually

released upon in vivo biodegradation of the

hydrogel itself53. We also evaluated the effect of

FGF-2-incorporated PCH on the wound healing

process using healing impaired diabetic db/db

mice (Figure 4)59. Our main conclusions were

that FGF-2-incorporated PCH show a

substantial effect to induce vascularization and

granulation tissue formation and improve wound

healing in the db/db mice59.



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Figure 4

Enhanced wound-healing in FGF-2-incorporated PCH-treated db/db mice.

To create a moist environment for wound

healing, alginate/chitosan/fucoidan

complexed hydrocolloids (ACF-HS) has been

developed as a functional wound

dressing21,22. ACF-HS gradually adsorbed

fluid without any maceration and the fluid

adsorbance in vitro reached constant during

18 hours. Round full-thickness skin defects

were made on the back of db/db mice to

prepare healing-impaired wounds22.

Application of ACF-HS could be expected

that it effectively interact with and protect

wound in rats, providing a good moist healing

environment with exudate. Besides those,

the wound dressing could have other

properties like ease of application and

removal, and proper adherence. After

applying ACF-HS to the wounds, the mice

were later killed and histological sections of

the wound were prepared. The histological

examinations have demonstrated

significantly advanced granulation tissue and

capillary formations in the wounds treated

with ACF-HS on day 4, day 9 and day 14, in

comparison with that in commercially

available hydrocolloid wound dressing

(ABSOCURE-surgical; Nitto Medical Corp.,

Osaka, Japan) as a positive control and

non-treatment (negative control)21,22.

Chitosan-based peptide/protein/gene

delivery systems

The design of appropriate carriers for an

administration of hydrophilic macromolecular

drug such as proteins and peptides has been

a major goal of pharmaceutical research.

Protein and peptide drugs are important to

treat diseases with increasing prevalence in

the population such as osteoporosis and

diabetes. On the other hand, vaccination



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(antigenic peptides) is also important and is

still a dire problem5. In general, drug delivery

materials can support via various routes, like

nasal, ocular, oral, parenteral and

transdermal. Particularly, protein and peptide

delivery by mucosal or oral routes would be

highly desirable from a clinical and industrial

perspective, and could lead to substantial

advances in the development and application

of proteins and peptides20,60,61. A series of

nanocarrier systems in which proteins and

peptides are associated with a chitosan

based nanostructure can be formed as

colloidal PECs1,5,6. PECs composed of

chitosan or its derivatives and proteins were

produced by mixing a protein solution with

chitosan solution. Hence, PEC comprised of

chitosan derivatives and insulin was

synthesized via electrostatic interactions62. A

second approach toward

protein/nanoparticles associations was to

make the colloidal PECs first and then to

adsorb the proteins, as reported by various

authors63. Since Mumper et al.64 pioneered to

apply chitosan to gene delivery systems, a lot

of efforts have been made to explore the

potential of chitosan and its derivatives as a

non-viral vector65-67. DNA/chitosan

complexes are prepared in acidic or neutral

aqueous solution where chitosan is highly or

partially ionized, respectively65-67. In addition

to solution pH, the DDAc and molecular

weight of chitosan influence the

physicochemical and biological properties of

chitosans and the transfection efficiencies of

DNA/chitosan complexes65-67. The use of

chitosan with more than 80% of DDAc might

accelerate chitosan degradation and DNA

release, since highly acetylated chitosan

(less than 20% of DDAc) release DNA very

slowly. Lavertu et al.66 studied several

combinations of various molecular weight

and DDAc value of chitosan, and they

selected two combinations of high

transfection efficiency using a chitosan of 10

kDa and DDAc of 8 and 20%. The coupling

between the DDAc and the molecular weight

of chitosan suggests that an optimal binding

strength of chitosan to DNA is required for

maximum transgene expression, namely, it

should be strong enough to condense and

protect DNA, but weak enough to permit

intracellular disassembly.

CONCLUSION

Chitin, a natural polymer of

N-acetylglucosamine, is the second-most

abundant polysaccharide in the nature after

cellulose, and is derived in the exoskeletons

of crustacean or shrimp, the cuticles of

insects and cell walls of fungi. Chitosan

comprising N-acetylglucosamine and

glucosamine can be obtained by alkaline

deacetylation of chitin and is found to be

nontoxic and biocompatible with living tissue.

Since chitosan can be hydrolyzed by

lysozyme, it is one of the biodegradable

polymers, and chitosan and the degraded

products are nontoxic, nonimunogenic and

noncarcinogenic. Chitosan hydrogel has

attracted considerable interest due to their

biological activities, that is, antimicrobiral,

antitumor, hypocholesrolemic functions and

stimulatory effect on wound healing.

Furthermore, there are enough scientific

evidences for the potentiality of chitosan

hydrogels in many medical applications such

as drug delivery carriers, tissue adhesives,

wound dressing, hematostats, scaffolds for

tissue engineering and protein/gene delivery

carriers.



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ACKNOWLEDGEMENT

All authors contributed to the conception,

writing, illustration, and revision of the

manuscript. The study was supported by the

Ministry of Education, Culture, Sports,

Science, and Technology of the Government

of Japan (grant no. 1058500).

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

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